Biochemical characterization of novel appendages containing DING/PstS family proteins

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Chapter 3 Cloning of DING/pstS genes from Pseudomonas strains⁴

Introduction

One of the objectives of this work was to create DING and PstS overexpression strains of P. aeruginosa and P. fluorescens SBW25, as well as gene-complemented strains of PA14 negative mutants in order to study their effects on the formation of appendages. The recombinant constructs of PA14 wild-type and negative mutants were also to be used to study the role of DING and PstS proteins for studies of adhesion and cytotoxicity with the intestinal epithelial cell line, IEC-18. Additionally, we also aimed to obtain purified DING and PstS recombinant proteins in order to understand if the pure protein by itself has the ability to form appendages in vitro and also to study the effect of protein alone when added directly to the IEC-18 cell line. For this work the primers were designed based on the putative DING and pstS gene sequences obtained from the Pseudomonas Genome database. P. aeruginosa PAO1 does not possess a DING gene but it contains two DING-like genes, lapA (PA0688) and lapB (PA0689) and so in this case PA0688 was cloned and over-expressed. The entire coding region including the signal sequence was cloned and restriction sites were added to the 5’ and 3’ ends in order to facilitate directional cloning. The genes were amplified from genomic DNA of P. aeruginosa and P. fluorescens strains and the amplified fragments were then ligated to Pseudomonas-specific shuttle vectors, pBBR1MCS-5 and pME6032. The expression of these genes was induced with IPTG. The nucleotides coding for a hexa histidine-tag (His-tag) were added on the 3’-terminal end of the DING and pstS gene sequences of the P. aeruginosa PA14 strain in order to purify them using affinity chromatography. The DING gene sequence from the MDR25 strain is not known yet, so attempts were also made to clone the DING gene from these strains.

• Some parts of this chapter have been used in manuscript preparation (unpublished).
  The strategy followed for cloning the genes was as follows:
• To amplify DING and pstS genes from various aeruginosa strains and P. fluorescens SBW25 using PCR.
• Ligation of amplified PCR product into pGEMT vector and introduction of plasmid-containing insert into the cloning host coli DH5α.
• Plasmid preparation and restriction digestion of correct pGEMT/DING and pGEMT/pstS clones and shuttle vectors pBBR1MCS-5 or pME6032. Ligation of DING/pstS inserts to the shuttle vectors (expression vectors) and introduction of plasmid/insert into the cloning host coli DH5α.
• Introduction of pBBR1MCS-5/DING or pstS and pME6032/DING or pstS plasmids into PA14 wild-type and mutants using the triparental mating protocol and into PfluSBW25 and PAO1 strains using the electroporation protocol as described in Materials & Methods (Section 2.7c).
• The Pseudomonas transformants were also used for expression and purification of DING and PstS proteins from aeruginosa PAO1.
• The Pseudomonas transformants were also used for transmission electron microscopy for identification of appendages, protein expression studies and for cultured intestinal epithelial cell culture assays (Chapter 4 and 6)

Results

aeruginosa strains PA14 and PAO1 and P. fluorescens SBW25 strain

The primers used for the amplification of DING and pstS genes from the above-mentioned strains are described in Appendix E (Table E.1). PCR amplification of DING and pstS genes was carried out as described in Materials & Methods (Section 2.5d), and gave a single band at about 1.2 kb for the DING gene (or lapA in case of PAO1) and 1.0 kb for the pstS gene in all the three strains of Pseudomonas, as shown in Figure 3.1 & 3.2. The sizes of the PCR products were in good agreement with the sizes reported in the Pseudomonas Genome database for each gene (Winsor et al., 2011).

Construction of overexpression strains of Pseudomonas and gene-complementation in Pseudomonas mutants

Overexpression and gene-complemented strains of Pseudomonas were successfully created as described in Materials & Methods. The list of the recombinant strains created in this study is mentioned in Appendix A. During cloning, we found that PA14 negative mutants that were supposed to be only gentamicin-resistant were also resistant to tetracycline, thus preventing us from creating gene-complemented strains of some of the negative mutants.

Small scale induction for checking the expression of DING and PstS proteins

Small scale inductions were carried out to check if the transformations worked. This method was chosen as isolation of plasmid DNA from Pseudomonas requires use of the cesium chloride density gradient method which is a laborious and time-consuming procedure. The inductions were carried out and the samples were prepared for analysis as described in Materials & Methods (Section 2.9). The identities of the expressed proteins from the clones were further confirmed through immunoblot analysis.

Overexpression of DING protein in PA14 wild-type bacteria

As shown in Figure 3.5, inductions in TSB with IPTG led to an increase in DING protein expression. As evident from the gel, expression was seen in both supernatant as well as lysate (Left Panel). The expression was further verified through immunoblot analysis (Right Panel). The immunoblot confirms that the induction is seen only in overexpression strains and not in the wild-type, which indicates that the expression is plasmid-driven

Overexpression of PstS protein in PA14 wild-type bacteria

SDS-PAGE followed by Coomassie staining (Figure 3.6A) clearly shows that, upon induction in TSB media, there was a slight increase in PstS protein expression. As evident from the gel, expression was seen in both supernatant as well as lysate. This was further confirmed using immunoblot analysis. Figure 3.6B shows the immunoblot of PstS overexpression in PA14 wild-type. As seen in the blot, it is clear that the induction is seen only in overexpression strains and not in the wild-type, which indicates that the expression is plasmid-driven. The removal of signal sequence might have resulted in a slightly lower band in the secreted fraction as compared to that of the lysate.

Expression and Purification of Pseudomonas aeruginosa

UCBPP PA14 DING and PstS proteins

Previous attempts in our laboratory to purify recombinant PA14 DING from E. coli resulted in several issues, including poor solubility due to protein misfolding, and low yields. We therefore decided to carry out the expression in its native host system, P. aeruginosa. The expression and purification of PA14 DING and PstS proteins was carried out in P. aeruginosa PAO1 instead of the PA14 strain because of the unavailability of PC2 facilities for handling large-scale cultures of a highly-virulent strain. P. aeruginosa PAO1 is found to be moderately virulent and the overall strain similarity between PA14 and PAO1 is very high which makes it a suitable candidate for these protein expression studies. The genes were cloned into the E. coli – Pseudomonas shuttle vector pBBR1MCS-5 and transformed into PAO1 wild-type as described previously in Materials & Methods (Section 2.7c). Addition of tags such as the His-tag, or the maltose binding protein (MBP) tag, at the C- or N- terminal has been widely used for ease of purification. Though these tags are found to enhance protein solubility they are sometimes found to interfere with proper protein folding (Young et al., 2012). As such we constructed overexpression strains with and without a His-tag at the C-terminal end. Due to the presence of signal sequence at the N-terminal end we chose to add the His-tag at the C-terminal end.

Chapter 1 General introduction 
1.1 Introduction to P. aeruginosa and P. fluorescens
1.2 The Gram-negative cell envelope
1.3 Overview of mechanisms of pathogenesis by P. aeruginosa
1.4 Quorum sensing (QS)
1.5 Biofilms
1.6 Protein secretion systems in Pseudomonas
1.7 Type II secretion system (T2SS) – the Xcp and Hxc systems and the formation of pseudopilin
1.8 OMVs as a novel protein secretion pathway in Gram-negative bacteria
1.9 Phosphate regulation in Gram-negative bacteria  Control of the Pho regulon
1.10 Influence of low phosphate on P. aeruginosa pathogenesis
1.11 PstS & DING – phosphate binding proteins in Pseudomonas spp
1.12 Aims of the current research
Chapter 2 Materials and Methods
2.1 Materials
2.2 Bioinformatics
2.3 Bacterial strains used in this study .
2.4 Vectors
2.5 Nucleic acid Methods
2.7 Cloning of DING and pstS genes into expression vectors pBBR1MCS-
2.8 Protein methods
2.10 Microscopy studies
2.11 Biochemical characterization of sheared appendages
2.12 Mammalian cell cultur
2.13 Sample preparation for Mass spectrometry studies
Chapter 3. Cloning of DING/pstS genes from Pseudomonas strains
3.1 Introduction
3.2 Results
3.3 Summary & Discussion
Chapter 4 Expression and localization of PstS/DING family proteins in clinical strains of Pseudomonas aeruginosa
4.1 Introduction
4.2 Results
4.3 Summary & Discussion
Chapter 5 Biochemical characterization of novel appendages containing DING/PstS family proteins
5.1 Introduction
5.2 Results
5.3 Summary & Discussion
Chapter 6 Effect of DING/PstS family proteins on intestinal epithelial cells
6.1 Introduction
6.2 Results
6.3 Summary & Discussion
Chapter 7 Summary and general discussion
7.1 DING and pstS gene cloning
7.2 Formation of novel appendages
7.3 Characterization of sheared appendages
7.4 Bacterial attack on epithelial cells
7.5 Protein structure and appendage formation
7.6 Are the appendages an extension of the outer membrane?
7.7 Other factors which influence appendage formation
7.8 In vitro and in vivo effects of DING, PstS and appendages
7.9 Additional lines of investigation in the future
7.10 Summary
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The roles of the phosphate-binding proteins as virulence factors of Pseudomonads